Thiopental Protects Human T Lymphocytes from Apoptosis in Vitro via the Expression of Heat Shock Protein 70

Article (PDF Available)inJournal of Pharmacology and Experimental Therapeutics 325(1):217-25 · May 2008with32 Reads
DOI: 10.1124/jpet.107.133108 · Source: PubMed
Abstract
Barbiturates, which are used for the treatment of intracranial hypertension after severe head injury, have been associated with anti-inflammatory side effects. Although all barbiturates inhibit T-cell function, only thiobarbiturates markedly reduce the activation of the transcription factor nuclear factor-kappaB (NF-kappaB). Various pharmacologic inhibitors of the NF-kappaB pathway are concomitant nonthermal inducers of the heat shock response (HSR), a cellular defense system that is associated with protection of cells and organs. We hypothesize that thiopental mediates cytoprotection by inducing the HSR. Human CD3(+) T lymphocytes were incubated with thiopental, pentobarbital, etomidate, ketamine, midazolam, or propofol. Human Jurkat T cells were transfected with small interfering RNA (siRNA) targeting heat 70-kDa shock protein (hsp 70) before thiopental incubation. Apoptosis was induced by staurosporine. DNA binding activity of HSF-1 was analyzed by electrophoretic mobility shift assay; mRNA expression of hsp27, -32, -70, and -90 was analyzed by Northern blot, and protein expression of hsp70 was analyzed by Western blot and flow cytometry after fluorescein isothiocyanate (FITC)-hsp70-antibody staining. Apoptosis was assessed by flow cytometry after annexin V-FITC or annexin V-phycoerythrin staining. Activity of caspase-3 was measured by fluorogenic caspase activity assay. Thiopental induced hsp27, -70, and -90 but not hsp32 mRNA expression as well as hsp70 protein expression. Thiopental dose-dependently activated the DNA binding activity of HSF-1, whereas other substances investigated had no effect. In addition, pretreatment with thiopental significantly attenuated staurosporine-induced apoptosis and caspase-like activity. Transfection with hsp70-siRNA before thiopental treatment reduced this attenuation. Thiopental specifically and differentially induces a heat shock response, and it mediates cytoprotection via the expression of hsp70 in human T lymphocytes.
Thiopental Protects Human T Lymphocytes from Apoptosis
in Vitro via the Expression of Heat Shock Protein 70
Martin Roesslein, David Schibilsky, Laurent Muller, Ulrich Goebel, Christian Schwer,
Matjaz Humar, Rene Schmidt, Klaus K. Geiger, Heike L. Pahl, Benedikt H. J. Pannen,
and Torsten Loop
Department of Anesthesiology and Critical Care Medicine, University Medical Center, Freiburg, Germany (M.R., D.S., L.M.,
U.G., C.S., M.H., R.S., K.K.G., H.L.P., T.L.); and Department of Anesthesiology and Critical Care Medicine, University Hospital,
Duesseldorf, Germany (B.H.J.P.)
Received October 16, 2007; accepted January 22, 2008
ABSTRACT
Barbiturates, which are used for the treatment of intracranial
hypertension after severe head injury, have been associated
with anti-inflammatory side effects. Although all barbiturates
inhibit T-cell function, only thiobarbiturates markedly reduce
the activation of the transcription factor nuclear factor-
B (NF-
B). Various pharmacologic inhibitors of the NF-
B pathway
are concomitant nonthermal inducers of the heat shock re-
sponse (HSR), a cellular defense system that is associated with
protection of cells and organs. We hypothesize that thiopental
mediates cytoprotection by inducing the HSR. Human CD3
T
lymphocytes were incubated with thiopental, pentobarbital,
etomidate, ketamine, midazolam, or propofol. Human Jurkat T
cells were transfected with small interfering RNA (siRNA) tar-
geting heat 70-kDa shock protein (hsp 70) before thiopental
incubation. Apoptosis was induced by staurosporine. DNA
binding activity of HSF-1 was analyzed by electrophoretic mo-
bility shift assay; mRNA expression of hsp27, -32, -70, and -90
was analyzed by Northern blot, and protein expression of
hsp70 was analyzed by Western blot and flow cytometry after
fluorescein isothiocyanate (FITC)-hsp70-antibody staining. Ap-
optosis was assessed by flow cytometry after annexin V-FITC
or annexin V-phycoerythrin staining. Activity of caspase-3 was
measured by fluorogenic caspase activity assay. Thiopental
induced hsp27, -70, and -90 but not hsp32 mRNA expression
as well as hsp70 protein expression. Thiopental dose-depen-
dently activated the DNA binding activity of HSF-1, whereas
other substances investigated had no effect. In addition, pre-
treatment with thiopental significantly attenuated staurospo-
rine-induced apoptosis and caspase-like activity. Transfection
with hsp70-siRNA before thiopental treatment reduced this
attenuation. Thiopental specifically and differentially induces a
heat shock response, and it mediates cytoprotection via the
expression of hsp70 in human T lymphocytes.
Barbiturates such as thiopental are frequently used for the
treatment of intracranial hypertension after severe head in-
jury. Although barbiturates exert protective effects in the
setting of focal neurological ischemia, their application has
also been associated with systemic anti-inflammatory side
effects (Drummond et al., 1995). With thiobarbiturates, these
effects may be mediated by the inhibition of important reg-
ulators of immune function, the transcription factors nuclear
factor of activated T cells, activator protein 1, and nuclear
factor-
B (NF-
B) (Loop et al., 2002, 2003; Humar et al.,
2004a,b).
Although the association between barbiturate therapy, de-
creased intracranial hypertension, and anti-inflammatory ef-
fects is clinically well documented, it is unclear whether the
protective actions reflect a specific effect of barbiturates.
Moreover, the underlying molecular mechanisms remain to
be identified.
The so-called heat shock response (HSR) is a cellular de-
fense system that is highly conserved throughout evolution
(Welch, 1992; Malhotra and Wong, 2002). It can be found in
a wide spectrum of organisms ranging from prokaryotes to
human beings. The underlying molecular mechanism is
based on the ability of the transcription factor heat shock
factor (HSF)-1 to display inducible DNA binding activity to
the consensus heat shock element (HSE) and involves mul-
tiple steps, including nuclear translocation, oligomerization,
M.R. and D.S. contributed equally to this work.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.107.133108.
ABBREVIATIONS: NF-
B, nuclear factor-
B; HSR, heat shock response; HSF, heat shock factor; HSE, heat shock element; hsp, heat shock
protein; hsp, heat 70-kDa shock protein; siRNA, small interfering RNA; FITC, fluorescein isothiocyanate; FL, fluorescence channel; PE,
phycoerythrin; 7-AAD, 7-amino-actinomycin D; I
B, inhibitor of nuclear factor-
B; ANOVA, analysis of variance; Hsc, heat shock cognate protein.
0022-3565/08/3251-217–225$20.00
T
HE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 325, No. 1
Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 133108/3322743
JPET 325:217–225, 2008 Printed in U.S.A.
217
at Physiologisches Inst der Univ Bibliothek on April 16, 2009 jpet.aspetjournals.orgDownloaded from
and inducible serine phosphorylation of HSF-1, the latter
being an important determinant of the transactivating po-
tency of HSF-1 (Westerheide and Morimoto, 2005). The HSR
is characterized by the expression of heat shock proteins
(hsps), which are synthesized by cells in response to heat,
hence the name, as well as to various other stressful stimuli
(De Maio, 1999; Kregel, 2002). Expression of hsps, which are
classified by their function and size, has been shown to pro-
tect cells from a broad range of cellular stressors, such as
hypoxia, oxygen radicals, endotoxin, infections, and fever (De
Maio, 1999). The cytoprotective capacity of heat shock pro-
teins may be attributed in part to their ability to stabilize
intracellular protein structures, which allows cells and or-
ganisms facing life-threatening insults resumption of normal
cellular and physiological activities (De Maio, 1999).
Several links have been established between the HSR and
the NF-
B pathway. The existing reciprocity between these
two pathways is underlined by the fact that various pharma-
cologic inhibitors of the NF-
B pathway, such as geldana-
mycin, dexamethasone, acetylsalicylic acid, and bimoclomol
are inducers of the HSR (Malhotra and Wong, 2002). In
contrast, induction of the HSR by either hyperthermia or
nonthermal inducers before a proinflammatory stimulus was
shown to inhibit subsequent inflammatory responses such as
the mononuclear cell expression of tumor necrosis factor-
and interleukin-1
(Schmidt and Abdulla, 1988; Snyder et
al., 1992).
We have recently reported that thiopental inhibits the
activation of NF-
B in human CD3
T cells (Loop et al., 2002,
2003). Therefore, the hypothesis of this study was to deter-
mine whether thiopental would also be able to mediate cyto-
protection in these cells by inducing the HSR.
Materials and Methods
Reagents. The following anesthetics were used: etomidate
(Braun, Melsungen, Germany), s-ketamine (Parke Davis, Berlin,
Germany), midazolam (Hoffman-La Roche, Grenzach-Wyhlen, Ger-
many), propofol (Astra-Zeneca, Plankstadt, Germany), and thiopen-
tal (Altana, Konstanz, Germany). The remaining reagents were
purchased from Sigma Chemie (Deisenhofen, Germany) unless spec-
ified otherwise.
Isolation of CD3
T Lymphocytes and Cell Culture. Periph-
eral blood mononuclear cells were isolated from blood bank product
buffy-coats obtained from healthy donors requiring no further Insti-
tutional Review Board consent. The cells were acquired by using
density centrifugation on Ficoll-Hypaque (GE Healthcare, Little
Chalfont, Buckinghamshire, UK) according to the manufacturer’s
recommendations. The cells were microscopically analyzed, and then
they were counted in a Neubauer chamber. For the isolation of CD3
T lymphocytes, the peripheral blood mononuclear cells (3– 4 10
8
)
were incubated for 15 min on ice with anti-CD3 antibodies conju-
gated to magnetic beats (Miltenyi Biotec, Bergisch-Gladbach, Ger-
many). Separation of CD3
cells was performed using an L/S column
(Miltenyi Biotec), and it was confirmed by flow cytometry (85%
purity).
Cell Culture and Transfection of Jurkat T Cells. Jurkat T
cells (ACC 282; DSMZ, Braunschweig, Germany) were cultured in
RPMI 1649 medium (Invitrogen, Karlsruhe, Germany) supple-
mented with 10% fetal bovine serum, 2 mM glutamine, and 100 U/ml
penicillin G and streptomycin (all obtained from Invitrogen, Carls-
bad, CA) each. Jurkat T cells in logarithmic growth phase were
washed in phosphate-buffered saline. Cells (2.5 10
6
–5 10
6
) were
transfected with 6
gofhspA1A small interfering RNA (siRNA)
directed against hsp70-mRNA or nonsilencing siRNA (both obtained
from QIAGEN GmbH, Hilden, Germany) according to the optimized
protocol for Jurkat T-cells using the Cell Line Nucleofector Kit V and
a Nucleofector II (Amaxa, Ko¨ln, Germany). After transfection, cells
were cultured in 5 ml of prewarmed medium and six-well tissue
culture plates for 18 to 20 h before treatment.
Total Cell Extracts and Electrophoretic Mobility Shift As-
says. Cells were harvested by centrifugation, they were washed once
in ice-cold phosphate-buffered saline, and then total cell extracts
were prepared as described previously (Loop et al., 2002). Total cell
extracts of CD3
T lymphocytes (1 10
7
cells/sample) were used for
electrophoretic mobility shift assays. Inhibitors of proteinases and
phosphatases were added at concentrations to the extraction and
suspension buffer as described previously (Loop et al., 2002). Band-
shift assays were performed using a
32
P-labeled HSF-1 oligonucleo-
tide (Promega, Madison, WI). The kinase reaction consisted of 37
l
of purified water, 25 ng of HSF-1 oligonucleotides, 5
l of kinase
buffer, 5
lof[
-
32
P]dATP (GE Healthcare), and 1.5
l of T4 kinase
(PNK buffer and PNK T4 kinase; New England Biolabs, Schwalbach,
Germany), and it was incubated for 30 min at 37°C. The protein
content of the cell lysates was determined using a Bradford assay
system (Bio-Rad Laboratories, Mu¨ nchen, Germany), and equal
amounts of protein (30
g) were added to a 20-
l electrophoretic
mobility shift assay reaction mixture containing 20
g of bovine
serum albumin, 2
g of poly(dI-dC) (Roche Diagnostics, Mannheim,
Germany), 2
l of buffer D (20 mM HEPES, pH 7.9, 20% glycerol,
100 mM KCl, 0.5 mM EDTA, 0.25% Nonidet P-40, 2 mM dithiothre-
itol, and 0.1% phenylmethylsulfonyl fluoride), 4
lof5 Ficoll buffer
(20% Ficoll 400, 100 mM HEPES, 300 mM KCl, 10 mM dithiothrei-
tol, and 0.1% phenylmethylsulfonyl fluoride),1
l of 50 mM MgCl
2
,3
l of double-distilled H
2
O, and 1
l of HSF-1
32
P-labeled oligonucle-
otide. These samples were incubated at room temperature for 30
min, and then they were loaded on a 4% acrylamide gel in 0.5 Tris
borate-EDTA (900 mM Tris-HCl, 900 mM boric acid, and 20 mM
EDTA, pH 8.0), 400
l of ammonium persulfate, and 40
l of tetra-
methylethylenediamine. For the supershift assays, 2.5
l of antibody
HSF-1 (clone 10H8, SPA-950E; Assay Designs, Ann Arbor, MI/
BIOMOL, Hamburg, Germany) or p65 (clone C20, SC-372X; Santa
Cruz Biotechnology, Heidelberg, Germany) were added to the reac-
tion simultaneously with the protein and incubated as described.
Gels were vacuum dried (Gel dryer 543; Bio-Rad, Hercules, CA) for
30 min, and then they were exposed to X-ray film (Kodak, Stuttgart,
Germany).
RNA Isolation and Northern Blot Analysis. Total RNA was
extracted from approximately 2 10
7
CD3
T lymphocytes/sample
according to the manufacturer’s recommendation (a monophasic one-
step solution of phenol and guanidine isothiocyanate; Invitrogen).
Aliquots of total RNA (10
g/lane) were size-fractionated on a dena-
turating 1% agarose gel, they were transferred to a nylon membrane
(Hybond-N; GE Healthcare) by capillary blotting in 20 sodium
saline citrate (3 M NaCl and 0.3 M sodium citrate), and then they
were cross-linked to the membrane by UV irradiation. The mem-
brane was preincubated for 30 min in hybridization solution (Ex-
pressHyb; Clontech, Palo Alto, CA), and then they were incubated
overnight at 68°C with a
32
P-labeled (Prime-It II labeling kit; Strat-
agene, La Jolla, CA) probe. The probes consisted of hsp27, hsp32,
hsp70, and hsp90 cDNA fragment. All blots were stripped and re-
probed with an 18S ribosomal RNA cDNA to confirm equal loading.
The following specific primers were used: hsp27, 5-ACGTCAAC-
CACTTCGCTCCTGAGG-3 as upper primer and 5-CTTGGCTC-
CAGACTGTTCCGAACTC-3 as lower primer; hsp70, 5-CAG
CGG CAG GCC ACC AAG GAC-3 as upper primer and 5-TGC
ACC GCC GCC CCG TAG G-3 as lower primer; hsp90, 5-GCG
AGT CGG ACG TGG TCC-3 as upper primer and 5-CTG AGG GTT
GGG GAT GAT GTC-3 as lower primer; and 18S, 5-CGC CGC
GCT CTA CCT TAC CTA CCT-3 as upper primer and 5-GAC
CGC CCG CCC GCT CCC AAG AT-3 as lower primer.
SDS-Polyacrylamide Gel Electrophoresis and Western
Blotting. Total cell extracts of 1 10
7
cells/sample were boiled in
218 Roesslein et al.
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Laemmli sample buffer, and then they were subjected to 10% SDS-
polyacrylamide gel electrophoresis as described previously (Loop
et al., 2002). Proteins were transferred to Immobilon P mem-
branes (Millipore Corporation, Eschborn, Germany). Equal load-
ing was confirmed by stripping membranes and incubating them
with specific antibodies at the end of the procedure. Primary
antibodies used for Western blotting were hsp70 (clone C92F3A-5,
SPA-810; Assay Designs/BIOMOL; dilution 1:1000), heat shock
cognate protein (Hsc)70 (polyclonal, SPA-816; Assay Designs/
BIOMOL; dilution 1:1000), HSF-1 (polyclonal, SPA-901; Assay
Designs/BIOMOL; dilution 1:1000), and
-actin (polyclonal, 4967;
Cell Signaling Technology Inc., Beverly; dilution 1:1000). Nonspe-
cific binding sites were blocked by immersing the membrane into
blocking solution [20 mM Tris-HCl, pH 7.6, 0.1% Tween 20 with
5% (w/v) nonfat dry milk powder (Fluka, Buchs, Switzerland)].
Membranes were washed in 20 mM Tris-HCl, pH 7.6, plus 0.1%
Tween 20, and then they were incubated in a recommended dilu-
tion of specific antibodies. Bound antibody was detected by goat
anti-rabbit/horseradish peroxidase-conjugated secondary anti-
body (7074; Cell Signaling Technology Inc.; dilution 1:2000). The
immunocomplexes were detected using enhanced chemilumines-
cence Western blotting reagents (GE Healthcare) according to the
manufacturer’s instructions. Exposure to enhanced chemilumi-
nescence Western blot films (GE Healthcare) was performed for
15 s to 1 min.
Fluorogenic Caspase Activity Assay. Total protein cell ex-
tracts of 1 10
7
cells per sample (10
l) were mixed with 90
lof
assay buffer (100 mM HEPES, pH 7.5, 2 mM dithiothreitol, and 2
mM phenylmethylsulfonyl fluoride). The respective fluorogenic sub-
strate for caspase-3 and -7, 7-amino-4-methylcoumarin (1
l, 60
M;
Alexis Corporation, Gruenberg, Germany), was added and the fluo-
rescence was measured at 30°C for 30 min in a Microplate Spectra
Max Gemini XS reader (Molecular Devices, Sunnyvale, CA) at 380/
460 nm.
Flow Cytometric Analysis of CD3
T Lymphocytes. After the
experimental treatment, CD3
T lymphocytes (1 10
5
cells/sample)
were harvested by centrifugation, they were washed in phosphate-
buffered saline, and then they were stained in annexin binding buffer
containing annexin V-fluorescein isothiocyanate (FITC) and propidium
iodide (all obtained from BD Biosciences, Heidelberg, Germany) or with
a monoclonal FITC-conjugated hsp70 antibody (clone C92F3A-5, SPA-
810F; Assay Designs/BIOMOL) using a 1:40 dilution for 30 min accord-
ing to the manufacturer’s recommendations. Subsequently, the percent-
age of early apoptotic lymphocytes was measured using a flow
cytometer (FACSCalibur; BD Biosciences). Lymphocytes were gated
using a forward/side scatter, and fluorescence intensity was measured
in 1 10
4
lymphocytes/sample in fluorescence channel (FL)1 for an-
nexin V and FL2 for propidium iodide.
Cell Staining and Multiparameter Flow Cytometry of Jur-
kat T Cells. Cells were harvested by centrifugation, they were
washed in phosphate-buffered saline, and then they were permeabil-
ized using the Cytofix/Cytoperm kit (BD Biosciences). The perme-
abilized cells were stained with a monoclonal FITC-conjugated hsp70
antibody (BD Biosciences) using a 1:40 dilution for 30 min. Trans-
fection efficiency of the used siRNAs estimated by Alexa Fluor 488-
labeled hspA1A-siRNA was 90% (data not shown).
For the detection of apoptosis and necrosis, cells were harvested by
centrifugation, they were washed in phosphate-buffered saline, and
then they were stained in annexin binding buffer containing annexin
V-phycoerythrin (PE) and 7-amino-actinomycin D (7-AAD) (all ob-
tained from BD Biosciences) at a 1:100 dilution according to the
manufacturer’s recommendation. Flow cytometry of 3 10
4
/sample
was performed with a Cyan cytometer (Dako Denmark A/S,
Glostrup, Denmark). Cells were gated using a forward/side scatter,
and fluorescence intensity was measured in FL2 for annexin V and
FL4 for 7-AAD.
Quantitative and Statistical Analysis. Differences in mea-
sured variables between the experimental conditions were assessed
using a one-way analysis of variance followed by a Student-Newman-
Keuls post-hoc test for multiple comparisons (normality test passed)
for caspase-3 activity data and a one-way analysis of variance on
ranks followed by a nonparametric Student-Newman-Keuls test for
multiple comparisons or Student’s t test for flow cytometric data.
Results were considered statistically significant at p 0.05. The
tests were performed using the SigmaStat software package (SPSS
Inc., Chicago, IL ).
Results
Thiopental Induces the Expression of Various Heat
Shock Proteins at the Transcriptional and Transla-
tional Level. To investigate whether thiopental induces hsp
expression, we performed Northern blot experiments to ex-
amine the effects of thiopental treatment on the mRNA levels
of hsp27, hsp32, hsp70, and hsp90 in T lymphocytes. Thio-
pental strongly increased the mRNA levels of hsp27 as well
as that of hsp70 and hsp90 in CD3
T lymphocytes (Fig. 1A,
blots 1, 3, and 4). In contrast, thiopental did not alter hsp32
mRNA levels (Fig. 1A, blot 2). Western blot analyses simi-
larly revealed hsp70 protein accumulation in response to
thiopental treatment (Fig. 1B). Flow cytometric analysis with
a monoclonal FITC-conjugated hsp70 antibody revealed
hsp70 expression in 50% CD3
T cells after thiopental
treatment (, p 0.05).
Thiopental Activates the DNA Binding Activity of
HSF-1 to the HSE. To assess whether the thiopental-medi-
ated induction of hsp gene expression was due to the activa-
tion of HSF-1/HSE DNA binding activity, we performed elec-
trophoretic gel mobility shift assays using a radiolabeled
HSE oligonucleotide as a probe. Although untreated control
cells displayed no induction (Fig. 2, lane 1), cells exposed to
thiopental for2hatincreasing doses showed moderate (100
g/ml) to strong (400 and 1000
g/ml) induction of the HSF-1
DNA binding activity (Fig. 2, lanes 3–5). Supershift analyses
using a specific antibody against an HSF-1 subunit and com-
petition analyses using unlabeled HSE oligonucleotides con-
firmed the specificity of the protein/DNA interaction (Fig. 2,
lanes 6–9).
Various Other Anesthetics Have No Effect on the
HSF-1/DNA Binding Activity. The results described above
raise the question whether the induction of HSF-1 activation
by thiopental is specific for this drug, or whether it may be a
common pharmacological effect of other anesthetic agents.
Therefore, we performed electrophoretic gel mobility shift
assays to test whether midazolam, propofol, etomidate, or
s-ketamine, when applied over a broad range of clinically
relevant concentrations, would also activate HSF-1. How-
ever, in contrast to thiopental, none of the other drugs in-
duced HSF-1 DNA binding activity in CD3
T lymphocytes
(Fig. 3A, lanes 2– 4; B, lanes 1–3; C, lanes 2–4; and D,
lanes 1–3).
Structurally Diverse Barbiturate Analogs Differ in
HSF-1 Induction. To test the hypothesis that the thio-group
is responsible for the thiopental-mediated effects, we com-
pared the ability of thiopental to induce HSF-1 to that of its
structural oxy-analog pentobarbital by electrophoretic gel
mobility shift assays. Incubation of CD3
T cells with thio-
pental (400, 1600, and 4100
M) induced the activation of
HSF-1, whereas equimolar concentrations of pentobarbital
did not (Fig. 4, lanes 2–4 versus lanes 7–9).
Thiopental Mediates Cytoprotection via Induction of hsp70 219
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Thiopental Leads to HSF-1 Phosphorylation. Because
inducible HSF-1 phosphorylation on serine residues is impor-
tant for HSF-1 activity, we examined the phosphorylation
state of thiopental-induced HSF-1 by Western blot. HSF-1
hyperphosphorylation was detected after treatment of CD3
T cells with thiopental (400
g/ml) after 1 and 2 h (Fig. 5,
lanes 3 and 5).
Thiopental Protects T Lymphocytes from Staurospo-
rine-Induced Caspase-3-Like Activity and Apoptosis.
We subsequently investigated whether thiopental-mediated
cytoprotection could be detected in this T-cell model by mea-
suring the activity of caspase-3, a pivotal enzyme in the
execution of apoptosis, using fluorogenic assay. Treatment of
CD3
T lymphocytes with the proapoptotic agent staurospor-
ine (2
M; 4 h) lead to a significant increase in caspase-3-like
activity (Fig. 6 A, column 2 versus column 1; , p 0.05). In
contrast, thiopental (100 or 400
g/ml; 8 h) alone had no
effect (Fig. 6A, bars 3 and 4). Preincubation with thiopental
at the same concentrations for 4 h before the addition of
staurosporine (2
M; 4 h) significantly reduced caspase-3-
like activity (Fig. 6A; bars 5 and 6 versus bar 2; , p 0.05).
Flow cytometric analysis using annexin V-FITC and pro-
pidium iodide staining of CD3
T lymphocytes was per-
Fig. 1. Effects of thiopental on the expression of various heat shock
proteins in human CD3
T lymphocytes. A, effect of thiopental treatment
for2hatdifferent concentrations on the mRNA levels of heat shock
proteins hsp27, hsp32, hsp70, and hsp90 (blots 1– 4). CD3
T lympho-
cytes (2 10
7
) were used per sample. mRNA levels were analyzed by
Northern blotting. Accuracy of loading and transfer was confirmed by
reprobing using a cDNA probe for 18s rRNA (blot 5). In blot 2, rat liver
after hemorrhagic shock served as positive control. B, hsp70 protein
accumulation in response to thiopental treatment. Protein levels were
analyzed by Western blotting. CD3
T lymphocytes (1 10
7
) were used
per sample. Accuracy of loading and transfer was confirmed by reprobing
using an antibody recognizing constitutively expressed Hsc70. The re-
sults are representative of four separate experiments. C, flow cytometric
determination of hsp70 protein accumulation in CD3
T lymphocytes
after thiopental treatment. CD3
T lymphocytes (1 10
5
) were used per
sample (, p 0.05 versus negative control, ANOVA, normality test
passed; n 8).
Fig. 2. Effects of thiopental on the DNA binding activity of HSF-1 in
human CD3
T lymphocytes. CD3
T lymphocytes (1 10
7
/sample) were
treated with thiopental for2hattheconcentrations indicated (lanes
3–5). Cells exposed to 42°C for 1 h served as positive control (lane 2).
Binding activity was assessed by electrophoretic mobility shift assay.
Supershift analyses using a specific antibody recognizing an HSF-1 sub-
unit and competition analyses using unlabeled HSE oligonucleotides
confirmed the specificity of the protein/DNA interaction (lanes 6 –9).
Position of the HSF-1/DNA complexes (Š). A nonspecific activity binding
to the probe (E); unbound oligonucleotide (). The results are represen-
tative of four separate experiments.
220 Roesslein et al.
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formed for the quantification of apoptosis and necrosis. As
shown in a representative experiment in Fig. 6B, staurospor-
ine (2
M; 4 h) induced a strong increase in the rate of
annexin V-positive cells (Fig. 6B, bottom left histogram; 41
versus 2% control), whereas thiopental alone had no influ-
ence on annexin V-FITC staining (Fig. 6B, top middle and
right histogram; 2 and 4%). Preincubation of the T cells with
thiopental (100 or 400
g/ml; 4 h) before staurosporine-treat-
ment (2
M; 4 h) lead to a substantial decrease in the rate of
annexin V-positive cells (Fig. 6B; 21%, bottom middle and
16%, bottom right histogram). The percentage of cells posi-
tive for both annexin V-FITC and propidium iodide, which
represent end stage apoptotic and necrotic cells, did not differ
significantly between treatments (Fig. 6C). Statistical anal-
ysis of six independent experiments revealed that the
changes were significant [thiopental-preincubated and stau-
rosporine-treated versus only staurosporine-treated T cells;
Fig. 6C; , p 0.05: 20% (14 –24%)* (100
g/ml) and 15%
(10–25%)* (400
g/ml) versus 39% (38–58%)].
Thiopental-Mediated Protection from Staurospor-
ine-Induced Apoptosis in Jurkat T Cells Is Mediated
by the Expression of hsp70. To evaluate the role of hsp70
in the thiopental-mediated protection from staurosporine-
induced apoptosis, we assessed whether the protective prop-
erties of thiopental would be altered after modifying the
expression level of this protein in response to thiopental. For
this purpose, Jurkat T cells were chosen as cell model and
transfected with siRNA directed against hsp70-mRNA before
Fig. 4. Effects of structurally diverse barbiturate analogs on the DNA
binding activity of HSF-1 in human CD3
T lymphocytes. CD3
T lym-
phocytes (1 10
7
/sample) were treated for 2 h with either thiopental
(lanes 2–4) or pentobarbital (lanes 7–9) at equimolar concentrations
indicated, corresponding to approximately 100, 400, and 1000
g/ml
thiopental. Cells exposed to 42°C for 1 h served as positive controls (lanes
5 and 10). Binding activity was assessed by electrophoretic mobility shift
assay. Position of the HSF-1/DNA complexes (Š). A nonspecific activity
binding to the probe (E); unbound oligonucleotide (). The results are
representative of four separate experiments.
Fig. 5. Effects of thiopental on the phosphorylation of HSF-1 in human
CD3
T lymphocytes. CD3
T lymphocytes (1 10
7
/sample) were treated
with thiopental for the durations and at the concentrations indicated
(lanes 2–7). Cells exposed to 42°C for 1 h served as positive control (lane
2). Protein levels were analyzed by Western blotting. Accuracy of loading
and transfer was confirmed by reprobing using an antibody recognizing
-actin. The results are representative of four separate experiments.
Fig. 3. Effects of various other anes-
thetics on the DNA binding activity of
HSF-1 in human CD3
T lympho-
cytes. CD3
T lymphocytes (1 10
7
/
sample) were treated for 2 h with ei-
ther midazolam (A, lanes 2–4),
propofol (B, lanes 1–3), etomidate (C,
lanes 2–4), or s-ketamine (D, lanes
1–3) at the concentrations indicated.
Cells exposed to 42°C for 1 h served as
positive controls (A, lane 5; B, lane 4;
C, lane 5; and D, lane 4). Binding ac-
tivity was assessed by electrophoretic
mobility shift assay. Position of the
HSF-1/DNA complexes (Š). A nonspe-
cific activity binding to the probe (E);
unbound oligonucleotide (). The re-
sults are representative of four sepa-
rate experiments.
Thiopental Mediates Cytoprotection via Induction of hsp70 221
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thiopental treatment. Flow cytometry experiments after
staining of cells with FITC-labeled hsp70 antibody revealed
that the percentage of cells positive for hsp70 significantly
increased after thiopental treatment (Fig. 7, column 2 versus
column 1; ⴱⴱ, p 0.001). However, the rate of hsp70-positive
cells was significantly reduced in cells initially transfected
with siRNA directed against hsp70-mRNA before thiopental
treatment (Fig. 7, column 3 versus column 2; ⴱⴱⴱ, p 0.001).
In contrast, initial transfection of cells with nonsilencing
RNA not targeting any known eukaryotic gene product left
the percentage of cells positive for hsp70 unaltered after
thiopental treatment (Fig. 7, column 4 versus column 2).
Subsequent flow cytometric analysis using annexin V-PE
and 7-AAD staining was performed for the detection and
quantification of apoptosis and necrosis. As shown in a rep-
resentative experiment in Fig. 8A, staurosporine (2
M;4h)
induced a strong increase in the rate of annexin V-positive
cells [Fig. 8A; histogram I; 62.5% (white-filled curve) versus
21.6% control (gray-filled curve)]. Preincubation of the T cells
with thiopental (100
g/ml; 4 h) before staurosporine-treat-
ment (2
M; 4 h) lead to a substantial decrease in the rate of
annexin V-positive cells [Fig. 8A; histogram II; 39.6% (white-
filled curve) versus 62.5% (gray-filled curve)]. Compared with
cells treated with thiopental before the addition of stauro-
Fig. 6. Effects of thiopental on
caspase-3-like activity (A) and exter-
nalization of phosphatidylserine (B
and C) after staurosporine-induced
apoptosis in human CD3
T lympho-
cytes. CD3
T lymphocytes were pre-
treated with thiopental for4hatthe
concentrations indicated before the
induction of apoptosis adding 2
M
staurosporine for an additional 4 h
(with thiopental still present). A,
caspase-3-like activity was assessed
using fluorogenic caspase activity as-
say. CD3
T lymphocytes (1 10
7
)
were used per sample. Relative fluo-
rescent units (RFU) denotes relative
fluorescent units (ⴱⴱⴱ, p 0.001 ver-
sus staurosporine, ANOVA, normality
test passed; n 8). B, representative
flow cytometric analysis after annexin
V-FITC staining. CD3
T lympho-
cytes (1 10
4
) were analyzed per
sample. C, statistical analysis of six
independent experiments (, p 0.05
versus staurosporine, ANOVA on
ranks, normality failed; n 6). PI,
propidium iodide.
222 Roesslein et al.
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sporine, initial transfection of cells with siRNA directed
against hsp70-mRNA before thiopental and subsequent stau-
rosporine treatment lead to a significant increase in the rate
of annexin V-positive cells [Fig. 8A; histogram III; 54.4% (white-
filled curve) versus 39.6% (gray-filled curve)], whereas transfec-
tion of nonsilencing siRNA did not [Fig. 8A; histogram IV;
29.2% (white-filled curve) versus 39.6% (gray-filled curve)]. Sta-
tistical analysis of five independent experiments revealed that
the changes were significant [Fig. 8B; 35.4 7.2% (thiopental
staurosporine, column 3) versus 62.4 9.2% (staurosporine,
column 2), ⴱⴱ, p 0.01; and 51.0 9.9% (hsp70 siRNA
thiopental staurosporine, column 4) versus 35.4 7.2% (thio-
pental staurosporine, column 3), , p 0.05].
The percentage of cells positive for 7-amino-actinomycin D,
which represent necrotic and end stage apoptotic cells, did
not differ significantly between the treatments or transfec-
tions of the cells (data not shown).
Discussion
Barbiturates may be beneficial in patients with severe
head injury and refractory intracranial hypertension. This
conclusion is based on a series of clinical studies showing
that administration of barbiturates can reduce intracranial
pressure and increase cerebral perfusion pressure after brain
trauma (Ghajar et al., 1995). However, little is known about
whether these beneficial effects are specific for barbiturates
and whether they are reflected by changes at the cellular
level.
The data presented here support the hypothesis that thio-
pental induces a heat shock response and that it mediates
cytoprotection at clinically relevant tissue concentrations by
several lines of evidence: 1) Thiopental-mediated heat shock
protein gene expression seems to be selective: whereas hsp32
was not induced at all, hsp27 was activated only weakly, and
hsp70 and hsp90 were strongly induced. 2) Thiopental-in-
duced hsp expression was dose-dependent. 3) The induction
of a HSR seems to be a specific characteristic of thiopental,
because other anesthetics tested over a broad range of clini-
cally relevant concentrations did not exert any effect on
HSF-1 activation. 4) Induction of HSF-1 DNA binding activ-
ity by thiopental is linked to inducible serine phosphoryla-
tion. 5) Thiopental exerts cytoprotective properties, because
induction of caspase-3-like activity and apoptosis by stauro-
sporine, a nonselective protein kinase inhibitor commonly
used to elicit apoptotic cell death, were attenuated after
pretreatment with thiopental. 6) hsp70 seems to be pivotal
for this protection, because the knockdown of its expression
suspends the thiopental-mediated cytoprotection.
Other i.v. and volatile anesthetics vary in their ability of
inducing one or several hsps. Xenon and isoflurane precon-
ditioning has been reported to induce cardiac hsp27, and
pentobarbital reduced the levels of hsp27 mRNA in aortic
smooth muscle cells, whereas propofol had no effect (Kozawa
et al., 2000). Exposure of rats to isoflurane has been shown to
induce mRNA, protein, and activity of hepatic hsp32 (Hoetzel
et al., 2002). However, neither hsp27 nor hsp70 mRNA could
be detected after6hofanesthesia with desflurane, sevoflu-
rane, or isoflurane (Hoetzel et al., 2002).
In eukaryotic cells, HSF-1 has been identified as the primary
stress-inducible molecule sensing environmental changes and
mediating heat shock gene expression (Westerheide and Mori-
moto, 2005). The data presented here provide evidence that
thiopental is able to induce a multistep process involving bind-
ing of HSF-1 and subsequent transactivation of heat shock
genes. It is noteworthy that this mechanism is similar to the
events that occur during hyperthermia, and it is quite different
from the activation by other drugs and stressors. For example,
despite its ability to bind to the promoter of the endogenous
hsp70 gene, indomethacin-induced HSF-1 is inert, and tran-
scription is not induced (Lee et al., 1995). In our study, thiopen-
tal was the only agent tested to induce the activation of HSF-1,
which is responsible for mediating the expression of several but
not all heat shock proteins. This observation, which is in accor-
dance with our study, has become evident when overexpression
of HSF-1 followed by heat shock experiments revealed that
HSF-1 repressed heme oxygenase-1 (hsp32) gene expression by
directly binding to the HSE of the heme oxygenase-1 gene
promoter (Chou et al., 2005).
Several studies have documented in vitro and in vivo in-
teractions between the induction of an HSR and the activa-
tion of NF-
B when both pathways are activated sequentially
(DeMeester et al., 2001; Malhotra and Wong, 2002). NF-
Bis
activated only after its inhibitory subunit I
B
has been
phosphorylated by I
B kinase. Own previous results suggest
that thiopental applied before an inflammatory stimulus is
an inhibitor of NF-
B, its trans-acting potency, and its down-
stream effects on immune cell function by altering the activ-
ity of I
B kinase, confirming previous in vitro studies (Wong
et al., 1997a,b; Loop et al., 2002, 2003; Malhotra and Wong,
2002). It is noteworthy that oxybarbiturates failed to inhibit
NF-
B in equimolar amounts in these investigations (Loop et
al., 2002, 2003). Because our previous data revealed that
Fig. 7. Effects of the transfection with hsp70 siRNA on the thiopental-
induced expression of hsp70 protein in Jurkat T lymphocytes. Jurkat T
lymphocytes were transfected with siRNA directed against hsp70-mRNA
(hsp70 siRNA) or nonsilencing siRNA not targeting any gene product.
After transfection, cells were treated with thiopental fo r4hatthe
concentration indicated. Expression of hsp70 was assessed by flow cytom-
etry after staining with a FITC-labeled antibody directed against hsp70
protein (HSP70-FITC-ab). Data represent the median, 10th, 25th, 75th,
and 90th percentile (ⴱⴱⴱ, p 0.001 versus control, ANOVA on ranks; ⴱⴱ,
p 0.01 versus thiopental, Mann-Whitney rank sum test; n 9).
Thiopental Mediates Cytoprotection via Induction of hsp70 223
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thiopental prevented the proteolysis of immunoreactive
I
B
, these findings suggest that the suppression of NF-
B
activation and HSR induction by thiopental may involve the
stabilization of I
B
, making it a point of interaction and a
putative novel hsp (DeMeester et al., 1997; Wong et al.,
1997a; Loop et al., 2002, 2003).
Our finding that thiopental induces an HSR and protects
from apoptotic cell death via hsp70 in this T cell model is
supported by reports about various heat shock proteins as
key determinants in the regulation of apoptosis (Xan-
thoudakis and Nicholson, 2000): hsp70, which functions as
molecular chaperone and confers protection against various
stressful stimuli in vitro (Wong et al., 1996, 1998) and in vivo,
such as ischemia/reperfusion injury (Marber et al., 1995), has
also been identified to inhibit cell death by preventing mito-
chondrial cytochrome c release and activation of procaspases
into caspases (Xanthoudakis and Nicholson, 2000). This hsp
also acts downstream of cytochrome c release and upstream
Fig. 8. Effects of the transfection with hsp70 siRNA on the
thiopental-mediated decrease from staurosporine-induced
externalization of phosphatidylserine in Jurkat T lympho-
cytes. Jurkat T lymphocytes were transfected with siRNA
directed against hsp70-mRNA (hsp70 siRNA) or nonsilenc-
ing siRNA not targeting any gene product. After transfec-
tion, cells were treated with thiopental for4hatthe
concentrations indicated before induction of apoptosis us-
ing staurosporine (2
M; 4 h). A, representative flow cyto-
metric analysis after annexin V-PE staining. Depicted are
four histogram overlays (I–IV), each demonstrating the
course of the number of cells positive for annexin V in
response to the interventions stated below each overlay in
normal (gray-filled curve) and underlined (white-filled
curve) writing. Accordingly, the percentage stated above
the marker line in each overlay corresponds to cells of the
gray-filled curve, whereas the underlined percentage
stated below the marker line corresponds to cells of the
white-filled curve, fulfilling the criterion of the marker.
Jurkat T lymphocytes (3 10
4
) were analyzed per sample.
ST, staurosporine; T100, thiopental, 100
g/ml. B, statis-
tical analysis of five independent experiments (mean
S.D.; ⴱⴱ, p 0.01 versus staurosporine; , p 0.05 versus
staurosporine thiopental; t test).
224 Roesslein et al.
at Physiologisches Inst der Univ Bibliothek on April 16, 2009 jpet.aspetjournals.orgDownloaded from
of the activation of caspase-3 (Xanthoudakis and Nicholson,
2000). In addition, hsp70 might be involved in preventing a
proposed caspase-independent cell death by suppressing c-
Jun NH
2
-terminal kinase activity (Gabai et al., 1997). Both
hsp70 and/or hsp90 can bind Apaf-1, a component of the
so-called apoptosome, thereby inhibiting its formation and
the activation of caspase-9 (Xanthoudakis and Nicholson,
2000). Whether hsps other than hsp70 are also involved in
the thiopental-mediated cytoprotection remains to be identi-
fied in future studies. Thiopental has recently been shown to
induce apoptosis in lymphocytes and Jurkat cells by a CD95-
independent mechanism and caspase-3 activation, which
seems to be in contrast to our results (Keel et al., 2005). Keel
and coworkers had also used freshly isolated human lympho-
cytes but incubated the cells with thiopental for 24 and 48 h,
considerably longer than treatment times in our model,
which were 4 h for the pretreatment and an additional 4 h
during which apoptosis was induced.
Although some possible explanations exist, the exact mech-
anism by which thiopental treatment leads to an HSR still
remains to be identified. That other thiol compounds have
been shown to act as nonthermal inducers of the HSR by
destabilizing or directly denaturating proteins suggests that
thiopental may elicit the HSR by a similar mechanism
(Senisterra et al., 1997). The relevance of the thiol group at
position C2 within the thiopental molecule is further sup-
ported by the fact that pentobarbital, the structural oxy-
analog of thiopental, failed to induce HSF-1 in equimolar
amounts.
In conclusion, the present study demonstrates that thio-
pental induces a differential HSR mediated by the activation
and phosphorylation of HSF-1 and that thiopental exerts
cytoprotective effects via the expression of hsp70. Thus, the
results of the present study provide a molecular rationale for
future investigations that systematically examine the organ-
specific induction of the HSR by thiopental.
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    • "However, a beneficial effect on neurological outcome is unproven and a critically discussed issue, mainly because of severe medical complications [36]. Although thiopental has been associated with inhibition of neuronal apoptosis [37], reduced excitotoxicity [38], [39], radical scavenging [40]–[42], and the induction of cytoprotective heat shock proteins [43], these experimental studies do not sufficiently explain major neuroprotective physiological observations such as decreased cerebral metabolism and reduced oxygen demand [44], [45]. "
    [Show abstract] [Hide abstract] ABSTRACT: Ischemic and traumatic brain injury is associated with increased risk for death and disability. The inhibition of penumbral tissue damage has been recognized as a target for therapeutic intervention, because cellular injury evolves progressively upon ATP-depletion and loss of ion homeostasis. In patients, thiopental is used to treat refractory intracranial hypertension by reducing intracranial pressure and cerebral metabolic demands; however, therapeutic benefits of thiopental-treatment are controversially discussed. In the present study we identified fundamental neuroprotective molecular mechanisms mediated by thiopental. Here we show that thiopental inhibits global protein synthesis, which preserves the intracellular energy metabolite content in oxygen-deprived human neuronal SK-N-SH cells or primary mouse cortical neurons and thus ameliorates hypoxic cell damage. Sensitivity to hypoxic damage was restored by pharmacologic repression of eukaryotic elongation factor 2 kinase. Translational inhibition was mediated by calcium influx, activation of the AMP-activated protein kinase, and inhibitory phosphorylation of eukaryotic elongation factor 2. Our results explain the reduction of cerebral metabolic demands during thiopental treatment. Cycloheximide also protected neurons from hypoxic cell death, indicating that translational inhibitors may generally reduce secondary brain injury. In conclusion our study demonstrates that therapeutic inhibition of global protein synthesis protects neurons from hypoxic damage by preserving energy balance in oxygen-deprived cells. Molecular evidence for thiopental-mediated neuroprotection favours a positive clinical evaluation of barbiturate treatment. The chemical structure of thiopental could represent a pharmacologically relevant scaffold for the development of new organ-protective compounds to ameliorate tissue damage when oxygen availability is limited.
    Full-text · Article · Oct 2013
    • "Interestingly, many cytoprotective proteins are preferentially synthesized during stress conditions associated with translational arrest (Yueh and Schneider, 2000; Hernandez et al., 2004). We observed that some carbimazole analogs induce HSP70, which was associated with cytoprotection (Roesslein et al., 2008). Because tissue repair is a late event following organ damage, we suggest first to minimize tissue damage by active pharmaceutical intervention, because the therapeutic restoration of neuronal tissue is unsolved. "
    [Show abstract] [Hide abstract] ABSTRACT: Oxygen deprivation during ischemic or hemorrhagic stroke results in ATP-depletion, loss of ion homeostasis, membrane depolarisation, and excitotoxicity. Pharmacologic restoration of cellular energy supply may offer a promising concept to reduce hypoxic cell injury. In this study we investigated whether carbimazole, a thionamide used to treat hyperthyroidism, reduces neuronal cell damage in oxygen-deprived human SK-N-SH cells or primary cortical neurons. Our results revealed that carbimazole induces an inhibitory phosphorylation of eukaryotic elongation factor eEF2 that was associated with a marked inhibition of global protein synthesis. Translational inhibition resulted in significant bioenergetic savings, preserving intracellular ATP-content in oxygen-deprived neuronal cells and diminishing hypoxic cellular damage. Phosphorylation of eEF2 was mediated by AMP-activated protein kinase and eEF2 kinase. Carbimazole also induced a moderate calcium influx and a transient cyclic adenosine monophosphate increase. To test whether translational inhibition generally diminishes hypoxic cell damage when ATP-availability is limiting, the translational repressors cycloheximide and anisomycin were used. Cycloheximide and anisomycin also preserved ATP-content in hypoxic SK-N-SH cells and significantly reduced hypoxic neuronal cell damage. Taken together, these data support a causal relation between the pharmacologic inhibition of global protein synthesis and efficient protection of neurons from ischemic damage by preservation of high-energy metabolites in oxygen-deprived cells. Furthermore, our results indicate that carbimazole or other translational inhibitors may be interesting candidates for the development of new organ-protective compounds. Their chemical structure may be used for computer-assisted drug design or screening of compounds to find new agents with the potential to diminish neuronal damage under ATP-limited conditions.
    Full-text · Article · Sep 2013
    • "b) Thiopental: is the prototype barbiturate used for anesthesia with an apoptotic mechanism by GABA-A agonistic action (64); also, through inducing lymphocyte death by "a CD95-independent mechanism" (65) and by "attenuated staurosporine-induced apoptosis and caspase-like activity" (66, 67); of course, the latter effect might be cardioprotective and against cardiomyocyte apoptosis. "
    [Show abstract] [Hide abstract] ABSTRACT: The modern practice of anesthesia is highly dependent ona group of anesthetic drugs which many of them are metabolized in the liver. The liver, of course, usually tolerates this burden. However, this is not always an unbroken rule. Anesthetic induced apoptosis has gained great concern during the last years; especially considering the neurologic system. However, we have evidence that there is some concern regarding their effects on the liver cells. Fortunately not all the anesthetics are blamed and even some could be used safely, based on the available evidence. Besides, there are some novel agents, yet under research, which could affect the future of anesthetic agents' fate regarding their hepatic effects.
    Full-text · Article · Aug 2013
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